Metabolomics of follicular fluid reveals the impact of heat stress on oocyte pick-up and in vitro embryo production efficiency in dairy cows.

Heat stress (HS) is a major challenge in global dairy farming, severely affecting reproductive performance. However, the molecular mechanisms by which it regulates oocyte development by altering the follicular microenvironment are unclear. This study assessed HS-related indicators in summer and autumn, dividing cows into HS and non-HS (TN) groups to evaluate the effect of HS on oocyte pick-up and in vitro embryo production (OPU-IVP) efficiency, and used untargeted metabolomics to analyze its impact on follicular fluid (FF) metabolic characteristics. The results showed that HS significantly affected the physiological and production performance of dairy cows. Serum and FF analysis showed significant changes in oxidative stress indicators (SOD, GSH-Px, CAT, MDA, T-AOC, ROS), inflammatory factors (IL-1, IL-6, TNF-α), heat shock proteins (HSP70 and HSP90), and steroid hormones (E2 and P4) induced by HS. Moreover, HS significantly reduced the total number of oocytes, oocyte recovery rate, proportions of grade A and B oocytes, cleavage rate, and blastocyst rate in OPU-IVP. Untargeted metabolomics analysis revealed 1544 differential metabolites in FF, which were mainly enriched in pathways such as steroid hormone biosynthesis, neuroactive ligand-receptor interactions, D-amino acid metabolism, tyrosine metabolism, phenylalanine metabolism, and tryptophan metabolism. In conclusion, this study not only reveals for the first time the metabolic characteristics of FF under HS and the potential mechanisms affecting oocyte development, but also provides innovative insights for developing precise interventions to improve OPU-IVP efficiency in dairy cows under HS.